SAN FRANCISCO—Though most of the work in CRISPR-Cas9 these days is finding new applications for the gene-editing system,
researchers out of the University of California, San Francisco (UCSF) have gone a different
route—and found a way to turn the system off. Their work, led by Dr. Benjamin Rauch—a postdoctoral researcher in the laboratory of Dr. Joseph
Bondy-Denomy, a UCSF Sandler Faculty Fellow in the Department of Microbiology and Immunology—was published in Cell Dec. 29, 2016 in a paper
titled “Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.”

Two of the biggest issues facing
CRISPR-Cas9 use for targeted gene editing, particularly in humans, is that it is generally not as precise as needed, with off-target edits occurring along
with the intended edits, and the concern that this method could cause harm intentionally or accidentally. This latest work out of UCSF could potentially
address both issues, according to Bondy-Denomy, in that it could offer more control and a fail-safe.

Most
laboratories working with CRISPR-Cas9 today use systems that feature a protein called SpyCas9 to “clip” DNA. In order to find bacteria with
inactive CRISPR systems, the team looked for “self-targeting” ones—strains of bacteria in which a virus had bypassed Cas9 and successfully
inserted its genes into the bacterium’s genome.

“Cas9 isn’t very smart,” Bondy-Denomy
explained. “It’s not able to avoid cutting the bacterium’s own DNA if it is programmed to do so. So we looked for strains of bacteria where
the CRISPR-Cas9 system ought to be targeting its own genome—the fact that the cells do not self-destruct was a clue that the whole CRISPR system was
inactivated.”

So the researchers used a bioinformatics approach designed by Rauch to study approximately 300
strains of Listeria, and discovered that 3 percent of the strains showed self-targeting and, within those, that there were four anti-CRISPR proteins
that could block the Listeria Cas9 protein, which is similar to SpyCas9. Of those four proteins, two of them—tagged as AcrIIA2 and
AcrIIA4—inhibited SpyCas9’s ability to target specific genes in other bacteria and in engineered human cells. As noted in their study,
“More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-
Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and
human cells.”

The increased control these anti-CRISPR proteins might help scientists increase or temporarily
block gene activity, or possibly synchronize “bursts” of activity from sets of genes, an approach that could offer answers about treating multi-
gene diseases. In addition, Bondy-Denomy noted that they also offer a way to stop any application of gene editing outside of the lab to prevent it from being
misused.

“The next step is to show in human cells that using these inhibitors can actually improve the
precision of gene editing by reducing off-target effects,” Rauch said. “We also want to understand exactly how the inhibitor proteins block
Cas9’s gene targeting abilities, and continue the search for more and better CRISPR inhibitors in other bacteria.”

This adds to the growing number of anti-CRISPR options researchers are uncovering. In late December, scientists from the University of Toronto and University of
Massachusetts Medical School shared news that they had discovered a trio of anti-CRISPRs, work that also appeared in Cell, in a paper titled
“Naturally Occurring Off-Switches for CRISPR-Cas9.” As the authors of the paper reported in their abstract, “Here, we report the discovery
of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins
bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-
CRISPR proteins now enable ‘off-switches’ for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome
editing in eukaryotes.” These families of anti-CRISPRs were found to inhibit N. meningitidis Cas9 both in vitro and in
vivo.

Broad core institute member Feng Zhang led the
study, which utilized a computational data-mining method to search microbial genomes for CRISPR systems, namely those that lack cas1, the most common gene
related to CRISPR immunity. The study was titled “Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory
Proteins Csx27 and Csx28,” and appeared in Cell.

“We establish that these CRISPR-Cas systems
can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its
own CRISPR array with short and long direct repeats, cleaves target RNA and exhibits collateral RNase activity … We also find that Csx27 represses,
whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and
monitor cellular transcripts,” the authors note in their abstract.

Cas13b has some similarities to Cas13a,
another RNA-targeting enzyme characterized by the Broad institute and colleagues in June 2016. Both enzymes only require a single guide RNA to specify the
target and are genetically encodable and capable of targeting multiple RNA transcripts at the same time. This newest Cas enzyme has some unique capabilities,
though, that are thought to make it a better option for improved targeting and manipulation of gene function.

“Beyond
direct RNA cleavage, there are numerous RNA-binding applications,” explained Aaron Smargon, graduate research assistant in the Zhang lab, who co-
authored the study with colleagues Neena Pyzocha and David Cox. “Transcriptomic RNA engineering will open up new avenues of research in biology,
particularly for complex dynamic systems such as those found in cancer, immunology and neuroscience.”